Extracellular matrix molecules, integrin receptors, cytoskeletal proteins, and regulators of these systems contribute to cell migration and signaling by complex, integrated mechanisms. We are addressing the following specific questions: 1. What biophysical phenomena and signaling mechanisms are important for efficient cell migration in two-dimensional and three-dimensional environments? 2. How are the functions of integrins, the extracellular matrix, and the cytoskeleton integrated, and how is the regulatory crosstalk between them coordinated to produce effective cell migration? We are using a variety of cell and molecular biology approaches to address these questions, including biochemical analyses, fluorescent protein chimeras, live-cell phase-contrast, confocal, and two-photon time-lapse microscopy, as well as methods for evaluating the local matrix responses to an individual migrating mammalian cell. We use a variety of fluorescent molecular chimeras and mutants of cytoskeletal proteins as part of a long-term program to analyze their functions in integrin-mediated processes. We have been focusing particularly on the functions and regulation of specific integrins and their associated extracellular and intracellular molecules in the mechanisms and spatial governance of cell migration. Normal, non-malignant cells are known to be able to sense and migrate toward regions of increasing stiffness. This phenomenon termed durotaxis is part of the normal mechanosensing capability of cells as they respond to the physical features of their local microenvironment as they migrate and maintain tissue homeostasis. We had previously developed an automated computer-based system for assessing the directional migratory properties of large populations of human cells on gradients of substrate stiffness. We used it to establish whether durotaxis depends on any specific migratory property, such as speed, or whether its efficiency varies depending on local stiffness. Unexpectedly, both non-malignant and all cancer cell lines we tested exhibited the strongest durotactic migratory response when located on quite soft regions of stiffness gradients (2-7 kPa). Cells located on stiffer regions showed progressively reduced responses to stiffness, but even cells on very stiff regions could still undergo durotaxis, albeit attenuated in effectiveness. The capacity for durotaxis was stable over time, i.e., independent of the length of time in culture. Interestingly, there was minimal correlation with the capacity of cells to migrate with directional persistence, as well as no correlation with speeds of cell migration. In terms of molecular mechanisms, the Arp2/3 molecular complex that mediates actin nucleation and branching is known to be a driver of leading-edge dynamics and cell migration. We found that inhibition of Arp2/3 function effectively impaired durotactic migration. Consequently, cells respond most effectively to gradients of stiffness in regions that are relatively soft, which results in their more rapid translocation from regions of matrix stiffness in the range of brain or breast tissue. A critical molecular component is Arp2/3, consistent with its key role in leading edge dynamics of migrating cells. Another series of studies of malignant versus non-malignant cells is focusing on the biophysical mechanisms of efficient cell migration in 3D collagenous matrices. These studies required developing methods for efficient fluorescent labeling of collagen without affecting its function. Using this methodology, the capacity of cells to apply forces to 3D matrix is being probed using particle image velocimetry (PIV) of confocal microscopy time-lapse movies of normal versus cancer cells. Normal cells apply strong pulling forces to the matrix at the front of migrating cells, resulting in pre-stressing or pre-tensioning of the matrix. This local modification of matrix suggests that cells stiffen the matrix in the direction of cell migration to provide effective formation and maintenance of cell adhesions. Both fibrosarcoma and metastatic breast cancer cells had defects in this phenomenon of anterior force generation, which was also observed after genetic ablation by CRISPR-Cas9 of myosin IIA in primary human fibroblasts. We had previously described a novel nuclear piston mode of migration in complex, crosslinked extracellular matrix in which myosin II actively pulls the cell nucleus forward to increase anterior intracellular hydrostatic pressure. A collaboration has established that the tropomyosin isoform 1.6 can positively regulate this intracellular pressure. This and prior research have separated the role of myosin II in nuclear piston migration from its known roles in regulating focal adhesions and cell-substrate traction. This combined approach involving characterization of the regulation of cell migration and phenotypes in various extracellular matrix microenvironments should provide novel approaches to understanding, preventing, or ameliorating migratory processes used by cells during abnormal embryonic development and particularly in cancer. An in-depth understanding of the precise manner by which cells move and interact with their matrix environment should also facilitate tissue engineering studies.